Micro-encapsulated aquaculture feed

ABSTRACT

To provide a feed that does not pollute rearing water, improves the immunity activity of  leptocephalus larvae,  is capable of directly feeding eel  leptocephalus,  and is capable of effectively inducing the growth of said larvae into glass eels. This micro-encapsulated aquaculture feed includes: an oil phase  11  having an oil-soluble nutrient component; a water phase  13  which is present inside the oil phase  11,  and which includes a water-soluble nutrient component; and a film  15  which includes the oil phase  11  and the water phase  13.  The water-soluble nutrient component includes at least one hydrolysate from among hydrolysates of amino acids, oligopeptides, and proteins.

TECHNICAL FIELD

The present invention relates to feed for aquaculture. Morespecifically, the present invention relates to feed for aquaculturecapable of effectively growing leptocephalus larvae of eels up to glasseels. The present invention relates to biotechnologically andimmunologically improved feed for aquaculture.

BACKGROUND ART

JP H11-253111 A (the following Patent Literature 1 (JP 2909536 B2))discloses an eel bait prepared by suspending shark egg powder inseawater. JP 2005-13116 A (the following Patent Literature 2 (JP 4530248B2)) discloses an eel bait containing a krill decomposition product or asoybean peptide with reduced phytic acid. The bait disclosed in JP2005-13116 A (the following Patent Literature 2) also basically containsshark eggs. JP 2011-239695 A (the following Patent Literature 3)discloses eel larvae feed containing Lampridae fish egg contents. JP2011-239696 A (the following Patent Literature 4) discloses eel larvaefeed containing fish egg contents with reduced protease activity.

JP H11-56257 A (the following Patent Document 5) discloses a bait forfeed organism of eel fry prepared by microencapsulating nutrientscontaining β-carotene. JP H11-56257 A (the following Patent Document 5)produces a microencapsulated bait for feed organism of eel frycontaining β-carotene by stirring and emulsifying β-carotene, gelatin,gum arabic and fish oil (paragraphs [0008], [0009] and [0013]).

JP 2012-505193 A (the following Patent Document 6) discloses animmunostimulator containing a microencapsulated cytokine. JP 2012-505193A (the following Patent Document 6) obtains microcapsules containingyeast expressing cytokines using malodextrin and a protective polymer(paragraph [0060]).

JP H10-327770 A (the following Patent Document 7) disclosesmicrocapsules for feed in which an aqueous phase containing awater-soluble nutrient component is present in an oil phase containingan oil-soluble nutrient component, including microcapsules coated with abiodegradable polymer film. This microcapsule is a W/O/W type emulsion.

CITATION LIST Patent Literature

Patent Literature 1: JP H11-253111 A

Patent Literature 2: JP 2005-13116 A

Patent Literature 3: JP 2011-239695 A

Patent Literature 4: JP 2011-239696 A

Patent Literature 5: JP H11-56257 A

Patent Literature 6: JP 2012-505193 A

Patent Literature 7: JP H10-327770 A

SUMMARY OF INVENTION Technical Problem

As described in Patent Literatures 1 to 4, feed based on shark eggs orLampridae fish eggs has been used for growing leptocephalus larvaecorresponding to the larval stage of Japanese eel (Anguilla japonica) ona small scale. However, feed based on fish eggs is in the form of paste,thus is dispersed in water. Therefore, when feed based on fish eggs isadministered to a large-scale culturing water tank, there is a problemthat not only bait efficiency is poor but also water in the culture tankis contaminated. For this reason, a bait that does not deteriorate waterquality even when culturing eels or the like on a large scale has beendesired.

Therefore, a bait using microcapsules has been developed as described inPatent Literature 5 above. However, these were not necessarily able toprevent deterioration of water quality, and in particular, it wasimpossible to effectively grow leptocephalus larvae of eels up to glasseels. In particular, the microcapsule of Patent Literature 5 is aso-called W/O type, and there is a problem that only oil-solublenutrient component can be contained as a nutrient component. For thisreason, the microcapsule feed in Patent Literature 5 is feed for baitorganisms, that is, mainly intended for use as enrichments for baitorganisms commonly used in seed production of fish culture, likerotifers such as Brachionus plicatilis, a brine shrimp, etc. Further,Patent Literature 5 uses a hardly digestible polymeric substance such asgelatin or gum arabic to obtain microcapsules. Since the microcapsulesin Patent Literature 5 are as small as 1 to 20 μm, there is apossibility that larval fish prey directly. However, larval fish withweak digestive function (for example, leptocephalus larvae) cannotcompletely digest this bait, thus there is a problem that utility valueas feed for larval fish is very low.

Patent Literature 6 describes a product obtained by encapsulating asuspension containing a cytokine-expressing yeast by a spray dryingmethod. The product of Patent Literature 6 is primarily intended tocertainly orally administer cytokines to fish culture, so a bait isseparately required. That is, the product of Patent Literature 6 is tobe mixed into fish culture feed and thrown.

An object of the present invention is to provide feed that does notcontaminate raising water and enhances the immune activity ofleptocephalus larvae, can be directly thrown to eel leptocephalus, andcan effectively grow the eel leptocephalus up to glass eels.

Solution to Problem

The present invention is basically based on the finding in an examplethat microencapsulated feed for aquaculture in which an aqueous phasecontaining a water-soluble nutrient component is present in an oil phasecontaining an oil-soluble nutrient component is feed for aquaculturesuitable also for mass culture, without contaminating water quality.

In addition, the present invention is based on the finding that, bycontaining an immunostimulator for stimulating immune activity,leptocephalus larvae of eels, which were difficult to grow up to glasseels, can grow extremely efficiently up to glass eels.

That is, a first aspect of the present invention relates tomicroencapsulated feed for aquaculture 17. The feed for aquaculture ismicroencapsulated feed for aquaculture containing an oil phase 11 havingan oil-soluble nutrient component, an aqueous phase 13 that is presentin the oil phase 11 and contains a water-soluble nutrient component, anda coating 15 containing the oil phase 11 and the aqueous phase 13.Moreover, the water-soluble nutrient component includes any one or moreof amino acids, oligopeptides, and protein hydrolysates. Thewater-soluble nutrient component may further contain saccharidesselected from any one or more of monosaccharides, oligosaccharides, andpolysaccharides.

Preferred feed for aquaculture of the present invention is feed forleptocephalus larvae of eels that is used for growing leptocephaluslarvae of eels up to glass eels.

Preferred feed for aquaculture of the present invention is one in whichthe water-soluble nutrient component contains a protein hydrolysate, andthe protein hydrolysate is obtained by hydrolyzing a protein sourcecontaining either or both of a vegetable protein and an animal protein,using a proteolytic enzyme, a hydrochloric acid, or hot water.

Preferred feed for aquaculture of the present invention is one in whichthe water-soluble nutrient component contains a protein hydrolysate, andthe protein hydrolysate contains any one or more of soybeanenzyme-treated proteins, fish and shellfish autolyzed extracts, fishmealenzyme-treated decomposition extracts, and fish meat hot water-treateddecomposition extracts.

Preferred feed for aquaculture of the present invention is one in whichthe coating 15 is a biodegradable polymer film.

Preferred feed for aquaculture of the present invention further containsan immunostimulator. Examples of the immunostimulator are any one ormore of lactic acid bacteria, yeasts, aspergillus oryzae, hay bacillus,Bacillus subtilis var natto, intestinal bacteria derived from adult fishintestines of fish of the order Anguilliformes, intestinal bacteriaderived from glass eel intestines of fish of the order Anguilliformes,and intestinal bacteria derived from leptocephalus larva intestines offish of the order Anguilliformes. By using feed for aquaculturecontaining these immunostimulators, it is possible to effectivelyproduce leptocephalus larvae of eels, which have been conventionallythought to be difficult to produce, up to glass eels.

Advantageous Effects of Invention

That is, according to the present invention, since nutrients aremicroencapsulated, it is possible to provide feed for aquaculture whichdoes not degrade (deteriorate) water quality even when culturing fish ona large scale, unlike a pasty fish egg bait. Further, according to thepresent invention, it is possible to provide feed for aquaculturecapable of effectively growing larval fish which is relatively difficultto produce seedlings, such as being able to enhance the immune activityof leptocephalus larvae and effectively grow to glass eels.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a conceptual diagram of the feed for aquaculture of thepresent invention.

FIG. 2 is a conceptual diagram of a manufacturing process in Example 1.

FIG. 3 is a photograph replacing the drawing when the microcapsulesobtained in examples are dispersed in distilled water.

DESCRIPTION OF EMBODIMENTS

Hereinafter, modes for carrying out the present invention will bedescribed using the drawings. The present invention is not limited tothe embodiments and examples described below, but also includes thoseappropriately modified within the scope obvious to those skilled in theart, based on the following embodiments.

The first aspect of the present invention relates to microencapsulatedfeed for aquaculture. Microencapsulation means to adjust feed such asbaits to a size which is easy for aquatic organisms to prey and thelike. The microencapsulated feed for aquaculture may have any shape suchas spherical shape, pellet shape, and elliptical shape, and the averagemaximum diameter is, for example, 1 nm or more and 1000 μm or less, andmay be 100 nm or more and 500 μm or less, 1 μm or more and 200 μm orless, or 5 μm or more and 100 μm or less. Particularly, when the feedfor aquaculture of the present invention is used as feed for larvaleels, a particle size of 1 to 200 μm is preferable. The feed for larvaleels is feed for growing leptocephalus larvae to glass eels. TheJapanese eels lay eggs near a water depth of about 200 meters in thespawning ground area, and the fertilized eggs hatch with graduallydecreasing water depth, and become larva shaped like a leafy leaf calledleptocephalus. When this leptocephalus grows and reaches the maximumelongation stage (6 cm or more), it undergoes metamorphosis,transforming from a flat body to a cylindrical body, and is said to bean almost transparent glass eel at a total length of about 6 cm.

Feed for Aquaculture

Feed for aquaculture means, for example, feed given as bait or nutrientsto aquatic organisms (aquatic animals). Feed for aquaculture can be usedas feed given to fish and shellfish in the aquaculture industry, and canalso be used as feed for aquarium fish or a ground bait for fishing. Thefeed for aquaculture of the present invention may be administered aloneas bait or nutrients, or may be administered mixed with other feed.

As described above, the fish targeted by the feed for aquaculture of thepresent invention is not particularly limited, but the feed foraquaculture of the present invention can be preferably used for fish ofthe order Anguilliformes and particularly preferably used for larvalfish (leptocephalus larvae) of the fish of the order Anguilliformes.Examples of such fish of the order Anguilliformes include Japanese eel(Anguilla japonica), European eel (A. anguilla), American eel (A.rostorata), giant mottled eel (A. marmorata), New Guinean eel (A.bicolor pacifica), Indonesian eel (A. bicolor bicolor), Mozambican eel(A. mossambica), Australian eel (A. australis australis), Australianfreshwater eel (A. australis schmidtii), Australian long-finned eel (A.reinhardtii), Cerebus eel (A. celebesensis), Polynesian long-finned eel(A. megastoma), Pacific short-finned eel (A. obscura), New Guineanalpine eel (A. interioris), Indian mottled eel (A. nebulosa), NewZealand long-finned eel (A. diffenbachii), Luzon eel (A. luzonensis),Bengali eel (A. bengalensis bengalensis), African eel (A. bengalensislabiata), continental freshwater eel (A. breviceps), continental eel (A.nigricans), Indonesian long-finned eel (A. malgumora), and the like.Other fish of the order Anguilliformes include common Japanese conger(Conger, myriaster), beach conger (C. japonica), Ariosoma meeki,Gnathophis nystromi nystoromi, Synaphobranchus kaupii, moray(Gymnothorax kidako), conger pike (Muraenesox cinereus), and pike eel(Muraenesox bagio).

The larval fish of the order Anguilliformes described in the presentinvention refers to the stage from the hatched larval fish to glass eelsthrough the leptocephalus larvae. Fry of fish of the orderAnguilliformes refers to the stage in which a glass eel has grown inboth internal and external shapes, and the stage called “Kuroko” inwhich a black pigment has been deposited in the external shape.

FIG. 1 is a conceptual diagram of the feed for aquaculture of thepresent invention. As shown in FIG. 1, the feed for aquaculture containsan oil phase 11 having an oil-soluble nutrient component; an aqueousphase 13 that is present in the oil phase 11 and contains awater-soluble nutrient component; and a coating 15 containing the oilphase 11 and the aqueous phase 13 therein. In the feed for aquacultureof the present invention, it is not necessary for the oil phase 11 andthe aqueous phase 13 to be completely separated, and all or a part ofthem may be in a mixed state. In particular, the aqueous phase may bedispersed in the oil phase.

The oil phase 11 may include, for example, one or both of animal oilsand vegetable oils. Examples of the animal oils are oils extracted fromfish eggs, fish oils, bird eggs (eg., chicken eggs), mammals and birds,and animal oils derived from fats and oils-producing bacteria. Examplesof the vegetable oils are soybean oil and corn oil.

Examples of the oil-soluble nutrient component are various animal fatsand oils, vegetable fats and oils, and fatty acids extracted andpurified from them. The oil-soluble nutrient components includeoil-soluble components as well as fat-soluble components. Other examplesof the oil-soluble nutrient component are oil-soluble vitamins (such asvitamins A, D, E) and carotenoids such as β-cryptoxanthin. Besides thosecontained in the above animal oils and vegetable oils, oil-solublevitamins themselves may be separately added to these oil-solublevitamins. Also, other examples of the oil-soluble nutrient component areDHA and EPA. The concentration of the oil-soluble nutrient component is,for example, 5 to 30 wt %, and preferably 10 to 20 wt %, of theoil-soluble liquid.

The aqueous phase 13 contains a water-soluble nutrient component.Examples of the water-soluble nutrient component are any one or more ofsaccharides, amino acids, oligopeptides, protein hydrolysates,water-soluble vitamins, pantothenic acid, and nicotinic acid. Examplesof the saccharides are monosaccharides, oligosaccharides, andpolysaccharides. Specific examples of the saccharides are glucose,1,5-anhydro-D-fructose, maltose, and trehalose. Leptocephalus larvae arelow in digestive ability, thus saccharides composed of small moleculessuch as monosaccharides and disaccharides are preferable as thesaccharides. In addition, since the feed of the present invention ismicroencapsulated feed having a structure of W/O/W, it is possible toeffectively store saccharides composed of small molecules and ingest itto leptocephalus larvae.

An example of the amino acid is essential amino acids. An oligopeptideis a peptide formed by binding 2 to several hundred (e.g., 300) aminoacids. An example of the protein hydrolysate is a hydrolysate using aprotein source containing either or both of a vegetable protein and ananimal protein using a proteolytic enzyme, hydrochloric acid, or hotwater. The protein hydrolysate may be, for example, any one or more ofsoybean enzyme-treated proteins, fish and shellfish autolyzed extracts,fishmeal enzyme-treated decomposition extracts, and fish meat hotwater-treated decomposition extracts.

An example of the vegetable protein is a soybean protein. Examples ofthe animal proteins are fish and shellfish extracts and zooplanktonextracts. The soybean enzyme-treated protein is a soybean proteinsubjected to enzyme treatment. An example of the enzyme is a protease(proteolytic enzyme). That is, the soybean enzyme-treated protein isobtained by reducing the molecular weight of soybean protein withenzyme.

The fish and shellfish autolyzed extract is an extract extracted fromfish and shellfish decomposed by autolysis. An example of a fish andshellfish autolyzed extraction method is described, for example, in JP3268657 B2. The fish and shellfish autolyzed extraction method maydecompose by a digestive enzyme contained in fish and shellfish itself,and decomposition may be promoted by, if necessary, adding an acid orhot water, and if necessary, acting a protease, shredding the fish andshellfish itself into mince, or by stirring. Examples of the fish andshellfish are anchovy, sardines, squid, and hill. The fish and shellfishautolyzed extract contains a lot of low molecular weight compounds suchas amino acids. It is preferable that the fish and shellfish autolyzedextract is an extraction of water-soluble parts from fish and shellfishdecomposed by autolysis. That is, fish and shellfish contain hardtissues such as bones and exoskeleton, and when a hard tissue is mixedin even a little bit, the digestive system of leptocephalus larvae thatincorporate it will be damaged. For this reason, the fish and shellfishautolyzed extract that extracts water-soluble parts of decompositionproducts can be preferably incorporated even the leptocephalus larvaewith weak digestive system.

The fishmeal enzyme-treated decomposition extract is an extract of oneobtained by decomposing a powdery product of fish and shellfish bypowder enzyme treatment. The method of enzyme treatment is described,for example, in JP 3408958 B2. In this method, fish and shellfish aretreated with a proteolytic enzyme under stirring to obtain an emulsifiedcomposition. This emulsified composition is formed from a liquid phasecontaining a water-soluble amino acid, oligopeptide and vitamin, andwater-soluble mineral components such as salts, and a solid phaseincluding a fat and oil containing a water-insoluble highly unsaturatedfatty acid and a protein having a molecular weight of 20,000 to 100,000.The emulsified composition is subjected to solid-liquid separation, andthe liquid portion may be extracted as a fishmeal enzyme-treateddecomposition extract. Other examples of the enzyme treatment are thosedescribed in JP 4804003 B2.

Fish meat hot water-treated decomposition extract is a method oftreating fish meat with hot water under pressure to decompose the fishmeat to obtain an extract. This hot water may properly contain aproteolytic enzyme. Specific example of hydrothermal treatment isdisclosed in WO 2002/036802 A.

Specific examples of the protein hydrolyzate are soybean peptides, fishand shellfish extracts, yeast extracts, and phytoplankton extracts.Examples of the water-soluble vitamins are vitamins B1, B2, B6, and C.Vitamin C is preferable because it also functions as an antioxidant. Anexample of the concentration of the water-soluble nutrient component is1 mg/ml to 500 mg/ml and preferably 2 mg/ml to 100 mg/ml, and may be 3mg/ml to 100 mg/ml in the aqueous solution. An example of the solutionis a buffer, and an example of the buffer is a phosphate buffer. Otherexamples of the solution are pure water, volume heavy water, saline, andphysiological saline.

Example of the weight ratio of the aqueous phase 13 to the oil phase 11is 1:10 to 10:1, and may be 1:5 to 5:1, or 1:3 to 3:1. Example of theweight ratio of the water-soluble nutrient component to the oil-solublenutrient component is 1:10 to 10:1, and may be 1:5 to 5:1, or 1:3 to3:1. Example of the weight ratio of the saccharides to the amino acidsource (amino acid, oligopeptide, protein hydrolysate) is 1:10 to 10:1,and may be 1:5 to 5:1, or 1:3 to 3:1.

The microcapsule feed of the present invention is formed by being coatedwith the coating of biodegradable polymer as the wall material, in thestate where the aqueous phase as described above is present in the oilphase. Examples of the biodegradable polymers used for the coating ofthe feed for aquaculture of the present invention are polysaccharidepolymers such as cellulose, polypeptides, nucleic acids, aliphaticpolyesters, and gelatin. Examples of the polysaccharide polymers arecellulose and polylactic acid polymers, among which polylactic acidpolymers are preferred. The number average molecular weight of thesepolymers is 1,600 or more and 460,000 or less, preferably 80,000 or moreand 160,000 or less, and may be 140,000 or more and 160,000 or less. Inparticular, when the feed for aquaculture of the present invention isadministered to leptocephalus larvae, the number average molecularweight of the polymer is preferably from 80,000 to 100,000. Thethickness of the coating of the feed for aquaculture of the presentinvention is, for example, 1 nm or more and 1 μm or less, and may be 1μm or more and 50 μm or less, and is preferably 10 nm or more and 10 μmor less.

In the microcapsule feed of the present invention, various substancesmay be added to the above aqueous phase or oil phase, or to the insideof the coating not mixed with the aqueous phase or oil phase. Examplesof such additives are algal components such as spirulina, driedspirulina, spirulina extract, chlorella, dried chlorella, and chlorellaextract. The algal component is contained, for example, in an amount of0.1 wt % or more and 10 wt % or less, and may be contained in an amountof 0.5 wt % or more and 5 wt %, in the microcapsule feed.

Preferred feed for aquaculture of the present invention further containsan immunostimulator. Examples of the immunostimulator are any one ormore of lactic acid bacteria, yeasts, aspergillus oryzae, hay bacillus,Bacillus subtilis var natto, intestinal bacteria derived from adult fishintestines of fish of the order Anguilliformes, intestinal bacteriaderived from glass eel intestines of fish of the order Anguilliformes,and intestinal bacteria derived from leptocephalus larva intestines offish of the order Anguilliformes. By using feed for aquaculturecontaining these immunostimulators, it is possible to effectivelyproduce leptocephalus larvae of fish of the order Anguilliformes, whichhave been conventionally thought to be difficult to produce, up to glasseels. The immunostimulator is contained, for example, in an amount of0.1 wt % or more and 10 w t% or less, and may be contained in an amountof 0.5 wt % or more and 5 wt %, in the microcapsule feed. As theintestinal bacteria derived from adult fish intestines of fish of theorder Anguilliformes, intestinal bacteria derived from glass eelintestines of fish of the order Anguilliformes, and intestinal bacteriaderived from leptocephalus larva intestines of fish of the orderAnguilliformes, for example, intestinal bacteria taken from theintestines of healthy adult fish of fish of the order Anguilliformes,glass eel of fish of the order Anguilliformes and leptocephalus of fishof the order Anguilliformes, and those obtained by culturing the takenintestinal bacteria can be used. In addition, these may use, forexample, bacteria taken after dissolving feces of healthy adult fish offish of the order Anguilliformes, glass eel of fish of the orderAnguilliformes and leptocephalus of fish of the order Anguilliformes,and those obtained by culturing the taken bacteria.

Method for Producing Feed for Aquaculture

The present invention also provides a method for producing themicroencapsulated feed for aquaculture of the present invention.

The feed for aquaculture of the present invention can be produced byproperly adopting the means used for encapsulation. The manufacturingprocess which is the basis of the feed for aquaculture of the presentinvention is, for example, as follows.

The manufacturing process includes a primary emulsification step, asecondary emulsification step, and an evaporation step. Next, each stepwill be described in detail.

Primary Emulsification Step

The primary emulsification step is a step of adding an aqueous solution(internal aqueous phase) of a water-soluble nutrient component to anoily solution (organic phase) in which a biodegradable polymer isdissolved in a (volatile) organic solvent as an oil-soluble nutrientcomponent and a wall material polymer and stirring the mixture to adjusta W/O type emulsion.

Examples of the organic solvents are volatile organic solutions such asalkyl halides, arylalkyls, and ethers. Preferred examples of thesolution of the oil-soluble liquid are dichloroethane, chloroform,toluene and dimethyl ether which are low-boiling organic solvents, amongwhich dichloroethane is preferred. In the primary emulsification step,in addition to the above components, the element used in a knownemulsification step may be appropriately added. For example, in theprimary emulsification step, a suitable emulsion stabilizer may beblended. Examples of such emulsion stabilizers include varioussurfactants, water-soluble resins, water-soluble polysaccharides and thelike generally used for emulsion adjustment, such as span-typesurfactants like sorbitan monoate. The amount of the surfactant is, forexample, 0.5 to 5 wt % of the oil-soluble liquid, and may be 1 to 3 wt %or 1 to 2 wt %.

In the primary emulsification step, an aqueous solution is obtained byputting the water-soluble nutrient component to a suitable solution. Atthat time, a protective material polymer is added for protecting thewater-soluble nutrient component including bacteria. Examples of theprotective material polymer include water-soluble polymericpolysaccharides like alginates and chitosan, and polyvinyl alcohols.Sodium alginate is particularly preferred. The water-solubleconcentration in the case of using sodium alginate is preferably 0.5 to5 wt %, and when it is too high, the dispersion stability of the W/Otype emulsion is lowered and aggregation tends to occur.

Then, a W/O type emulsion can be obtained by using an emulsifyingmachine (homogenizer), or gradually injecting an aqueous solution intothe prepared oily solution while stirring. The ratio (volume ratio) ofthe aqueous solution to the oily solution is, for example, 1:1 to 1:10,and may be 1:2 to 1:10, or 1:2 to 1:5. The primary emulsification stepis preferably carried out under ice cooling, and the temperature of theoily solution is, for example, −15° C. to 4° C., and may be −10° C. to0° C. The stirring speed is, for example, 1,000 to 10,000 rpm, andpreferably 3,000 to 5,000 rpm. Stirring may be performed by ultrasonicvibration. The stirring time is, for example, from 10 minutes or more to1 hour or less, and may be from 10 to 20 minutes.

Secondary Emulsification Step

The secondary emulsification step is a step of adding the W/O typeemulsion obtained in the above primary emulsification step to an aqueoussolution (external aqueous phase: second aqueous solution) differentfrom one used in the primary emulsification step, and stirring themixture to adjust a W/O/W type emulsion.

The second aqueous solution is preferably a solution containing acoating material or a dispersion stabilizer. Examples of the secondaqueous solution are pure water, distilled water, and physiologicalsaline. This second aqueous solution (external aqueous phase) is anaqueous solution of a water-soluble dispersion stabilizer. Examples ofthe water-soluble dispersion stabilizer include sodium polyacrylate,polyacrylamide, polyethylene imine, polyethylene oxide, polyvinylpyrrolidone, and the like. Polyvinyl alcohol is particularly preferred.It is also recommended to include at least tricalcium phosphate forsuppressing aggregation of droplet particles. Distilled water is used toadjust an aqueous solution of about 1 to 30 wt %, and particularlypreferably 2 to 10 wt %.

The secondary emulsification step is preferably carried out at a slowerstirring speed and in a shorter time than in the primary emulsificationstep. That is, a W/O type emulsion may be added to and mixed with anaqueous phase to be an external aqueous phase at ordinary temperature,and by continuing at a stirring speed of 300 to 1000 rpm for about 3 to10 minutes, drop coalescence of the internal aqueous phase is carriedout in each particle of the W/O type emulsion droplet that is adispersed phase. By this droplet coalescence, the W/O type emulsiondroplet is added to a droplet of a structure in which the inner singleaqueous phase is covered with the outer organic phase.

Evaporation Step

The evaporation step is a step of evaporating the organic solvent fromthe W/O/W type emulsion obtained in the secondary emulsification step,thereby forming microcapsules including the aqueous phase containing thewater-soluble nutrient component in the oil phase containing theoil-soluble nutrient component.

After the above droplet coalescence, one or both of warming anddecompression is performed under stirring, in order to volatilize andremove the low-boiling organic solution of the organic phase byin-liquid drying. It is recommended to simultaneously perform warmingand decompression from the viewpoint of processing efficiency. Thisevaporation step performs gentle stirring while warming to a temperatureslightly higher than the boiling point of the volatile solution. Whenthe low-boiling organic solvent of the organic phase is mainly composedof dichloroethane, the maximum achieving temperature is about 35° C. andthe maximum pressure reduction is about 300 hPa, in in-liquid dryingwhich simultaneously performs warming and decompression.

The stirring speed in in-liquid drying is preferably about 100 to 1000rpm, and the step time is preferably 1 to 24 hours, and particularlypreferably 3 to 10 hours. It is preferable that the obtainedmicrocapsules are filtered and dried and then cryopreserved, or filteredand then stored in an aqueous phase.

In each of the above steps, necessary nutrient components andimmunostimulators may be appropriately mixed.

Aquaculture Method Using Feed for Aquaculture

The present invention also provides a culture method using themicroencapsulated feed for aquaculture of the present invention.

As a method for culturing fish and shellfish, a known method may beappropriately adopted. Particularly, when culturing eel fry, forexample, the apparatus disclosed in JP 2013-236598 A may be used. Thisapparatus is an apparatus for raising eels which raises eels underatmospheric pressure to induce sexual maturation. Moreover, thisapparatus includes a water tank for storing raising water and eels, awater supply unit for supplying raising water to the water tank, adrainage water unit for discharging the raising water from the watertank, and a unit for adjusting the concentration of dissolved oxygen inthe raising water.

When raising eel larvae using the feed of the present invention, it ispreferable to directly put the above feed in a water tank for raisingeel larvae and feed it in the precipitated or dispersed state. Whencirculating water in a raising water tank, it is preferable to stop orintermittent the water flow while feeding, in order to suppress the lossof bait in the drainage. It is preferable to feed so that the feedalways remains and does not run short, and feed it from 1 to 5 separatetimes per day.

Example 1

Hereinafter, the present invention will be specifically described withreference to examples. The present invention is not limited by theexamples, and those appropriately adopting known methods are alsoincluded in the present invention. Microcapsules suitable as feed ofcultured larval fish were produced according to the above-describedproduction method of microcapsules, using the following compositions andconditions. FIG. 2 is a conceptual diagram of a manufacturing process inExample 1.

Adjustment of Internal Aqueous Phase

36 ml of a phosphate buffer solution was added to and mixed with soybeanenzyme-treated protein (manufactured by FUJI OIL CO., LTD.) and maltose,so as to be 20 mg/ml and 1 mg/ml, respectively, 1 ml of krillenzyme-treated decomposition extracting solution was further added, and1 wt % sodium alginate was further added to adjust an internal aqueousphase.

Adjustment of Organic Phase

15 wt % Feed oil (manufactured by SANSHO BUSSAN CO.,LTD.), 5 wt %polylactic acid polymer (average molecular weight 100,000) and 1.5 wt %sorbitan monooleate were added to and mixed with 108 ml ofdichloroethane to adjust an organic phase.

Adjustment of External Aqueous Phase

4 wt % of polyvinyl alcohol and 0.3 wt % of tricalcium phosphate wereadded to and mixed with 680 ml of distilled water to adjust an externalaqueous phase.

The internal aqueous phase was added and mixed while stirring theorganic phase under ice cooling at 5000 rpm for 10 minutes to adjust aW/O type emulsion, and the W/O type emulsion was added to the externalaqueous phase under stirring at ordinary temperature (20° C.) to adjusta W/O/W emulsion, followed by stirring at 150 rpm for 30 minutes atatmospheric pressure. Thereby, droplets in the internal aqueous phase inthe dispersed W/O type emulsion droplet are united, then the resultingdroplets were subjected to in-liquid drying treatment at a liquidtemperature of 35° C. for 6 hours under an atmospheric pressure of 300hPa, the produced microcapsules were separated by filtration, washedwith 0.1 molar concentration of hydrochloric acid aqueous solution toremove the tricalcium phosphate, and further washed with distilledwater, to recover the microcapsules.

The particle size of the microcapsules obtained using the aboveoperating conditions and compositions was measured with a laserdiffraction type particle size distribution apparatus, and found to havea particle diameter of 5 to 20 μm.

Example 2

Capsule feed was prepared in the same manner as in Example 1, exceptthat 1 ml of a commercially available enzyme that activatesmicroorganisms (LOVE Ibusuki) added with Bacillus subtilis var natto,dry yeast and lactic acid bacteria was added to the internal aqueousphase solution.

Example 3

Capsule feed was prepared in the same manner as in Example 1, exceptthat 1 g of fine powder of satsuma orange dried peel containing a largeamount of β-cryptoxanthin was added as a base to the phosphate buffersolution, the mixture was stirred at a low speed for 10 minutes,followed by centrifugation at 100 rpm for 5 minutes, and 1 ml of theobtained supernatant was added to the internal aqueous phase solution.

Example 4

Capsule feed was prepared in the same manner as in Example 1, exceptthat spiny dogfish eggs were used instead of the feed oil in Example 1.

Example 5

Capsule feed was prepared in the same manner as in Example 1, exceptthat 1 ml of spirulina extract was added to the internal aqueous phasesolution.

Example 6

Capsule feed was prepared in the same manner as in Example 1, exceptthat 1,5-anhydro-D-fructose was used instead of maltose.

Comparative Example 1

A bait was prepared using the method disclosed in JP H11-56257 (PatentLiterature 3). More specifically, β-carotene, gelatin and fish oil werestirred and emulsified to produce microencapsulated bait of eel frycontaining β-carotene (feed of Comparative Example 1).

Example 7

Using the feed of Examples 1 to 6 and Comparative Example 1, 20 each of7-day-old leptocephalus larvae were stored in a 100 ml-small glasscontainer, and a feeding test of the above samples was conducted. Witheach feed, contents were found in the alimentary canal of larvae, andfeeding was confirmed.

Example 8

Using the feed of Examples 1 to 6 and Comparative Example 1, 200 each of7-day-old leptocephalus larvae were stored in a 5 1-bowl shaped watertank, and a survival test using the above samples was performed. Withthe eel fry bait (Comparative Example 1) disclosed in JP H11-56257(Patent Literature 5), the survival rate remarkably decreased after thestart of the test, but with the feed of the present invention, thesurvival rate showed a result equivalent to the feed for larvae mainlyconsisting of shark eggs so far. Further, in the feed of the presentinvention, the water quality in the water tank was maintained.

Example 9

Adjustment of Aqueous Phase

16 g of soybean peptide and 1 g of sodium alginate were added to 80 mlof distilled water to adjust an aqueous phase.

Adjustment of Oil Phase

1.3 g of span 80 ((Z)-9-octadecenoic acid) was mixed to 130 g ofrapeseed oil to adjust an organic phase.

Adjustment of Added Layer W/O

30 g of rapeseed oil, 0.3 g of Span 80, 10 ml of distilled water and 0.7g of calcium chloride were mixed to adjust the added layer W/O.

The aqueous phase was added and mixed for 10 minutes while stirring theoil phase at 25° C. at 200 rpm, to adjust a W/O type emulsion. Addedlayer W/O was added and mixed for 5 minutes while stirring this W/O typeemulsion at 25° C. at 500 rpm. Further, it was stirred at 25° C. at 200rpm to accelerate the polymerization reaction for 45 minutes. Filtrationwas performed to collect 20 g of microcapsules. 20 g of distilled waterwas added to and dispersed in 20 g of the collected microcapsules, and aphotograph was taken. The obtained photograph is shown in FIG. 3. Thatis, FIG. 3 is a photograph replacing the drawing when the microcapsulesobtained in Examples are dispersed in distilled water.

INDUSTRIAL APPLICABILITY

The present invention can be utilized particularly in the fisheriesindustry.

REFERENCE SIGNS LIST

-   11 Oil phase-   13 Aqueous phase-   15 Coating-   17 Feed for aquaculture

1. Microencapsulated feed for aquaculture, comprising: an oil phase (11)having an oil-soluble nutrient component; an aqueous phase (13) that ispresent in the oil phase (11) and contains a water-soluble nutrientcomponent; and a coating (15) containing the oil phase (11) and theaqueous phase (13), wherein the water-soluble nutrient componentcontains any one or more of amino acids, oligopeptides, and proteinhydrolysates.
 2. The feed for aquaculture according to claim 1, whereinthe water-soluble nutrient component further comprises saccharidesselected from any one or more of monosaccharides, oligosaccharides, andpolysaccharides.
 3. The feed for aquaculture according to claim 1, whichis feed for leptocephalus larvae of eels, that is used for growingleptocephalus larvae of eels up to glass eels.
 4. The feed foraquaculture according to claim 1, wherein the water-soluble nutrientcomponent comprises a protein hydrolysate, and the protein hydrolysateis obtained by hydrolyzing a protein source containing either or both ofa vegetable protein and an animal protein, using a proteolytic enzyme, ahydrochloric acid, or hot water.
 5. The feed for aquaculture accordingto claim 1, wherein the water-soluble nutrient component comprises aprotein hydrolysate, and the protein hydrolysate contains any one ormore of soybean enzyme-treated proteins, fish and shellfish autolyzedextracts, fishmeal enzyme-treated decomposition extracts and fish meathot water-treated decomposition extracts.
 6. The feed for aquacultureaccording to claim 1, wherein the coating (15) is a biodegradablepolymer film.
 7. The feed for aquaculture according to claim 1, furthercomprising an immunostimulator.
 8. The feed for aquaculture according toclaim 7, wherein the immunostimulator comprises any one or more oflactic acid bacteria, yeasts, aspergillus oryzae, hay bacillus, Bacillussubtilis var natto, intestinal bacteria derived from adult fishintestines of fish of the order Anguilliformes, intestinal bacteriaderived from glass eel intestines of fish of the order Anguilliformes,and intestinal bacteria derived from leptocephalus larva intestines offish of the order Anguilliformes.